Specificity and Efficiency of NaV1.8 Gene Silencing for Neuropathy Treatment

Gene silencing by RNAi has been widely used in experimental settings and as a potential therapeutic strategy for many diseases. The tetrodotoxin-resistant sodium channel 1.8 (NaV1.8) is one isoform of voltage-gated sodium channels, which contributes to pathogenesis of neuropathic pain. Efficient NaV1.8 gene knockdown with RNAi technology appears to alleviate pain symptoms in experimental neuropathy. However, off-target effects due to low level of RNAi specificity is a major concern that has not been addressed. OBJECTIVE: To test for knockdown efficiency and specificity of five different siRNA sequences targeting NaV1.8. METHODS: We constructed lentiviruses carrying short hairpin RNAs (shRNAs) with green fluorescence protein (GFP) targeted four specific sites of NaV1.8 mRNA (1203-1233, 3407-3425, 6009-6027 & 6033-6051) and one scrambled sequence. These constructs were transduced into primary sensory neurons isolated from rat dorsal root ganglia (DRG). shRNA-mediated gene knock-down efficiency was examined via flow cytometry and immunocytochemistry; quantitative real-time PCR also examined selectivity by assessing mRNA levels of five NaV isoforms (1.3, 1.6, 1.7, 1.8 & 1.9). RESULTS: Flow cytometry showed 80% transduction efficiency of primary cells transduced with this lentivirus. Real-time PCR revealed NaV1.8 shRNA targeting 6033-6051 showed not only 50% (p=0.009, n=6) knockdown of NaV1.8 but also 30% (p=0.025, n=5) off-target knockdown of NaV1.6 mRNA. NaV1.8 shRNA against 1203-1233 showed 20% (p=0.036, n=6) off-target knockdown of NaV1.3 but was ineffective against NaV1.8. NaV1.8 shRNA targeting 6009-6027 selectively decreased NaV1.8 mRNA by 40% (p=0.033, n=6). Two days after transduction of scrambled siRNA, NaV1.8 shRNA (6009-6027) and untransduced neurons, immunocytochemistry with a NaV1.8 C-terminal peptide antibody showed obvious decreases in NaV1.8 protein in DRG neurons treated with 6009-6027 shRNA. CONCLUSION: These findings demonstrate that even though RNAi is a promising approach for gene therapy, neglecting to determine its specificity may cause off-target effects or failure to silence disease-associated genes.